Additive manufacturing of embedded materials

ABSTRACT

In one aspect, a method includes providing support material within which the structure is fabricated, depositing, into the support material, structure material to form the fabricated structure, and removing the support material to release the fabricated structure from the support material. The provided support material is stationary at an applied stress level below a threshold stress level and flows at an applied stress level at or above the threshold stress level during fabrication of the structure. The provided support material is configured to mechanically support at least a portion of the structure and to prevent deformation of the structure during the fabrication of the structure. The deposited structure material is suspended in the support material at a location where the structure material is deposited. The structure material comprises a fluid that transitions to a solid or semi-solid state after deposition of the structure material.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. §119(e)to provisional U.S. Patent Application No. 61/958,484, filed on Jul. 29,2013, the entire contents of which are hereby incorporated by reference.

FIELD OF THE USE

The present disclosure relates to fabricating multi-dimensionalstructures.

BACKGROUND

Additive manufacturing (AM) of biological systems has the potential torevolutionize the engineering of soft structures, bioprosthetics, andscaffolds for tissue repair. While 3D printing of metals, plastics, andceramics has radically changed many fields, including medical devices,applying these same techniques for the printing of complex and softbiological structures has been limited. The major challenges are (i)deposition of soft materials with elastic moduli of less than 100kilopascal (kPa), (ii) supporting these soft structures as they areprinted so they do not collapse, (iii) removing any support materialthat is used, and (iv) keeping cells alive during this whole processusing aqueous environments that are pH, ionic, temperature, andsterility controlled within tight tolerances. Expensive bioprinters thatattempt to address these challenges have been produced, but have yet toachieve results using soft hydrogels that are comparable to resultsachieved using commercial grade thermoplastic printers.

Some hydrogels are impossible to deposit in layers due to their tendencyto flow or deform under steady-state loading. However, hydrogels aredesirable materials for advanced biofabrication techniques because theirstructure underlies the function of complex biological systems, such ashuman tissue. 3D tissue printing (i.e., AM of tissues) seeks tofabricate macroscopic living composites of biomolecules and cells withrelevant anatomical structure, which gives rise to the higher-orderfunctions of nutrient transport, molecular signaling, and othertissue-specific physiology. Replicating the complex structures oftissues with AM requires true freeform fabrication, as tissues possessinterpenetrating networks of tubes, membranes, and protein fibers thatare difficult to fabricate using free-standing fused-deposition orphotopolymerization techniques. Conventional AM techniques may notpossess the level of spatial control necessary for freeform fabricationand rapid prototyping of soft tissues.

Recent advances in 3D tissue printing represent solutions to highlyspecific problems encountered in the AM of hydrogel materials, and areoften limited to a specific application. For example, Fused DepositionModeling (FDM) has been used to print avascular replicas ofcartilaginous tissues as well as fugitive vasculatures, which can beused to cast a vascularized tissue. Similar to the powders used in SolidFreeform Fabrication (SFF), dynamic support materials have beendeveloped to enable the fabrication of soft materials in complex spatialpatterns without the need of printed supports. These semi-solidmaterials may be capable of supporting the fusion of cells and gels;however, the latter cases are limited and do not constitute truefreeform fabrication. Indeed, the most successful methods forfabricating macroscopic biological structures in vitro rely on castingand not AM, as conventional AM techniques may not be sufficient torecreate true tissue complexity.

Many gels are ideal materials for biofabrication, because theirstructures underlie the function of complex biological systems, such ashuman tissues. The geometries of tissues may be difficult to recreatewithout techniques like Additive Manufacturing/3D printing, but themethods for 3D printing gels are limited. Many gels start as fluids andcannot be 3D printed without supports to prevent them from drooping oroozing. Conventional 3D printing techniques may not possess the level ofcontrol necessary for geometrically unrestrained 3D printing of gels andtissues. Attempts to print gels with FDM have yielded cartilage-liketissues as well as gels with simple networks of vessels, yet the resultshave been limited. Indeed, it is still easier and more effective to casta tissue than it is to 3D print it, as conventional 3D printingtechniques may not be sufficiently capable.

SUMMARY

The present disclosure describes a method for 3D printing gels referredto as Embedded Fusion Modeling (EFM), or Freeform Reversible Embeddingof Suspended Hydrogels (FRESH). EFM is similar to FDM, but instead ofdepositing a material on top of previous depositions or supports, EFMembeds material near other embedded deposits inside an omnipresentsupport bath and relies on the triggered assembly or reorganization ofthe material using targeted heating, photopolymerization, crosslinking,slow reaction kinetics, or application of binders. For example, thesupport bath may provide divalent cations for crosslinking the printedmaterial. For 3D printing techniques such as FDM, support materials areusually as stiff as the printed material and placed underneath orneighboring the print layers to prevent deformations. In EFM, thesupport material is everywhere, and the deposit is printed inside thesupport. The support bath is a non-newtonian fluid that allows fordeposition of new material while maintaining a buoyant, physical supportfor already embedded deposits. When two embedded deposits are closeenough, they fuse. After printing, the deposit can be removed from thesupport as a fully assembled construct.

In one aspect of the present disclosure, a method includes providingsupport material within which the structure is fabricated, depositing,into the support material, structure material to form the fabricatedstructure, and removing the support material to release the fabricatedstructure from the support material. The provided support material isstationary at an applied stress level below a threshold stress level andflows at an applied stress level at or above the threshold stress levelduring fabrication of the structure. The provided support material isconfigured to mechanically support at least a portion of the structureand to prevent deformation of the structure during the fabrication ofthe structure. The deposited structure material is suspended in thesupport material at a location where the structure material isdeposited. The structure material comprises a fluid that transitions toa solid or semi-solid state after deposition of the structure material.

Implementations of the disclosure may include one or more of thefollowing features. The support material may include a gel material. Thesupport material may include a hydrogel material. The support materialmay include micronized particulates. The support material may include athermo-reversible material. The structure material may include amaterial having an elastic moduli of less than 1 megapascal. Thestructure material may include at least one of a gel material that isdifferent from the support material, a metal material, a ceramicmaterial, or a polymer material. The structure material may include atleast one of an alginate material, a collagen material, a fibrinmaterial, a hyaluronic acid material, a protein material, apolysaccharide hydrogel material, synthetic gel material, an elastomerpolymer material, a rigid polymer material, or a polydimethylsiloxane(PDMS) elastomer. The structure material may be deposited using asyringe-based extruder that is inserted into the support material andextrudes the structure material into the support material. The methodmay include treating the structure material to cause the structurematerial to transition from the fluid to the solid or semi-solid stateafter the deposition of the structure material. The support material mayinclude a crosslinking agent for treating the structure material tocause the structure material to transition from the fluid to the solidor semi-solid state after the deposition of the structure material. Thecrosslinking agent may include at least one of calcium chloride orthrombin. The support material may include a material having a differentpH from the structure material for treating the structure material tocause the structure material to transition from the fluid to the solidor semi-solid state after the deposition of the structure material.Treating the structure material may include heating the structurematerial, cooling the structure material, or radiating the structurematerial with ultraviolet light. Removing the support material mayinclude heating the support material, cooling the support material, orremoving cations to disrupt crosslinking of the support material. Thefabricated structure may be a biological tissue or a tissue engineeringscaffold. Depositing, into the support material, the structure materialto form the fabricated structure can include depositing the structurematerial layer by layer in an XY plane, depositing the structurematerial layer by layer in an XZ plane, or depositing the structurematerial in a non-planar configuration.

In another aspect of the present disclosure, a method includesperforming a printing operation at approximately twenty degrees Celsius.The printing operation may include providing a gelatin slurry supportbath within which the tissue scaffold is fabricated, extruding, from anozzle, a hydrogel into the gelatin slurry support bath to form thetissue scaffold, and placing the gelatin slurry support bath with theextruded hydrogel into an incubator that is heated to approximatelythirty-seven degrees Celsius to melt the gelatin slurry support bath andto release the tissue scaffold. The hydrogel has an elastic moduli ofless than 1,000 kilopascal. The gelatin support bath flows in a presenceof an applied force and transforms into a non-flowing fluid in anabsence of the applied force. The gelatin slurry support bath includescalcium chloride to provide divalent cations to crosslink the hydrogelas the hydrogel is extruded out of the nozzle. The gelatin slurrysupport bath is configured to mechanically support the hydrogel and toprevent deformation of the hydrogel during printing of the tissuescaffold.

Implementations of the disclosure may include one or more of thefollowing features. The hydrogel may include at least one of alginate,collagen type I, fibrin, or hyaluronic acid. The hydrogel may include acell laden hydrogel.

Particular implementations of the subject matter described in thisdisclosure may be implemented to realize one or more of the followingpotential advantages. The 3D printing techniques described in thisdisclosure are applicable to gelling polymers and enable true freeformprinting of complex geometries. The techniques can be used to produceanatomically correct, perfusable scaffolds of coronary arteries. Thetechniques are cross-platform and can easily be adopted for use on manyopen-source and proprietary 3D printers.

Details of one or more implementations are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description, the drawings, and theclaims.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a 3D CAD rendering of a syringe-pump extruder.

FIG. 2 shows printing of a soft hydrogel using a support bath.

FIGS. 3A and 3B show printing using a 3D printer including an XYsyringe-based extruder that deposits hydrogels from a nozzle into athermo-reversible support bath.

FIG. 4A shows a 3D CAD model of a coronary artery vasculature from ahuman heart.

FIG. 4B shows a scaled-down print of the vasculature embedded within asupport bath.

FIG. 4C shows the printed vasculature after thermal release from thesupport bath.

FIG. 5 shows a printed alginate mesh of collapsed hexagonal unitsexhibiting a negative Poisson's ration behavior when transitioning froman unstrained form to a strained form.

FIG. 6 shows a parametric tubular structure that was designed in CAD andprinted.

FIG. 7 shows a cross-section view, a side view, and a close-upcross-section view of a two-material cylinder.

FIG. 8 shows a fluorescence image of an alginate filament embedded amonggelatin particles.

FIG. 9 shows a rectilinear pattern of filaments and fluorescence imagesand of the filaments at a scale.

FIG. 10 shows an octagonal pattern of filaments and fluorescence imagesand of the filaments.

FIG. 11 shows a helix being printed at various time points (a)-(c)

FIG. 12 shows 3D printed polydimethylsiloxane in the form ofinterconnected chain links.

FIG. 13 shows an image and a 3D representation of C2C12 cells printed inCollagen I gel.

FIG. 14 shows 3D representations of C2C12 myoblasts in a collagenconstruct.

FIG. 15 shows 3D representations of MC3T3 fibroblasts in a collagenconstruct.

FIG. 16 is a flowchart of a process for fabricating a multidimensionalstructure.

FIG. 17 shows a FRESH print of an embryonic chicken heart.

FIG. 18 illustrates weaving used to regenerate various muscle structuresusing the living muscle fiber threads.

DETAILED DESCRIPTION

Although the techniques described in the present disclosure areapplicable to a wide range of manufacturing processing methods, variousimplementations will be described below in the context of additivemanufacturing. While specific implementations are described, otherimplementations may exist that include operations and componentsdifferent than those illustrated and described below.

The present disclosure describes a method of embedding fused materials(EFM) within a support bath, where the embedded material is initially afluid or flowable material that transitions to a solid or semi-solidstate after deposition. The method can be used in the process ofadditive manufacturing (AM), also commonly referred to as freeformfabrication or 3D printing, of soft materials to fabricate 2D or 3Dstructures and objects based on a 3D digital design, which may bedifficult using traditional techniques or current additive manufacturingapproaches. While specific examples of soft materials and theircharacteristics are provided below, EFM is not limited to printingmaterials with a specific material hardness and other materials withdifferent characteristics may be printed.

The soft materials can include fluids that have substantially lowelastic modulus when being printed. Traditionally, printing a fluid inair may be difficult because the fluid may flow away from the depositionsite.

The soft materials can include solid materials that have an elasticmodulus in the range of approximately 10 GPa to 0.1 kPa. These solidmaterials may sag or deform if printed in air. For example, stiffmaterials such as cements, resins, and concretes can be printed usingEFM. Composites involving metal-ceramic or metal-carbon alloys can alsobe printed using EFM.

The soft materials can include materials that have an elastic modulusthat is initially low and increases over time due to crosslinking orassembly of the material. The final elastic modulus of these materialsmay be in the GPa range after crosslinking or assembly, but may bedifficult to print using other techniques because the elastic moduluswas much lower during the print process. An example of such materialsinclude an epoxy resin that is mixed but has not yet cured, so it is aliquid during printing but then cures overtime into a rigid plastic.Another example is a polydimethylsiloxane (PDMS) elastomer (e.g.,Sylgard 184, Dow Corning) that can be mixed as a prepolymer, printed asa liquid, and then cured for 48 hours at room temperature while stillimmobilized in the gelatin slurry support material before being releasedat 37 degrees Celsius.

The soft materials are printed inside a temporary support material thatcan be removed later by, e.g., heating or cooling the support materialto dissolve or melt the support material, or removing cations to disruptcrosslinking of the support material. Additional techniques for removingthe support material include vibration, irradiation with ultraviolet,infrared, or visible light, or application of a constant or oscillatingelectric or magnetic field.

The support material may be any material that acts as a viscoplasticmaterial with Bingham plastic-like rheological behavior. The supportmaterial may demonstrate a significant shear thinning behavior such thatthe support material acts like a solid material during deposition of thestructure materials and then acts like a fluid when the nozzle is movedthrough the support material such that the nozzle movement does notdisturb the deposited structure material. The support material mayexhibit viscoplastic behavior where it acts as a solid below a thresholdshear stress and flows like a liquid above the threshold shear stress.The characteristic that makes a support material suitable for EFM is adrop in viscosity under dynamic loading. In EFM, the dynamic loading iscaused by the force of an extruder moving through the support material,affecting the support material in a number of ways. The extruder couldchange the support material by imposing a mechanical load via shear,pressure, or vibration. The extruder could irradiate or heat the supportmaterial to thin it. Alternatively, a suitable material could loseviscosity under vibration, heating, or irradiation that occurs locallyto the extruder.

For example, the support material can include a Bingham plastic, orBingham plastic-like, material that is a solid material when notperturbed, but shear thins and provides minimal resistance when a nozzlemoves through it. The support material can include other materials withviscoplastic behavior, such as Herschel-Bulkley fluid. Bingham plasticsand Herschel-Bulkley fluids are viscoplastic materials included in the“shear-thinning” or “yield-stress fluid” category. Below a specificshear stress, these materials appear as a solid material. Above athreshold shear force, these materials behave as a fluid. A Binghamplastic may not necessarily “shear thin,” but rather may act much like aNewtonian fluid once it begins to flow. In contrast, theHerschel-Buckley fluid undergoes shear thinning once it begins to flow.

Thus, a 3D bioprinter can lower a syringe-based extruder into thesupport material and move around and deposit material in arbitrary 3Dgeometries. The extruded material stays in place once the tip of theextruder moves away, thus forming the 3D printed object. Once thecomplete 3D object is printed and the structural material hassufficiently assembled, the support material is removed.

A structure can be printed in any direction in 3D space. In addition tothe typical 3D printing that is done layer by layer in the XY plane, astructure can also be printed layer by layer in a non-XY plane, such asthe XZ plane, or a plane at any angle. A structure can also be printedin a non-planar fashion in a curved path, such as a helix. Structureswith material mechanical properties that are different in the plane ofprinting versus orthogonal to the plane of printing can thus be printedusing EFM. In particular, the fabricated structured can havethree-dimensions, and the fabricated structure can have printedanisotropic mechanical properties in a direction that lie in XY plane.For example, EFM can be used to print in the direction of nerve fibersin 3D, or in the direction of muscle fibers in 3D.

Some AM techniques rely on the triggered assembly or reorganization of amaterial using targeted heating, photopolymerization, or jetted glues tobind a powder substrate. EFM is more similar to Fused DepositionModeling (FDM), but in FDM, material is deposited on top of a previouslydeposited layer, which provides the necessary mechanical support tobuild multiple layers since this process occurs in an environment withno buoyant or conformal supports and requires the co-printing ofsupporting structures. In contrast, EFM deposits material nearpreviously deposited material, but not necessarily on top of it.Specifically, the support bath material provides mechanical support withthe deposited, embedded materials able to fuse in any direction as longas proximity is sufficient. To accomplish this, fusible material isdeposited into the support bath material, which behaves as a buoyant,non-Newtonian support. Support materials are usually as stiff as theintended deposit and placed underneath or neighboring the deposit toprevent deformation of the deposit. In EFM, the support material iseverywhere, and the deposit is embedded inside the support materialduring printing. After printing, the object can be removed from thesupport as an intact object.

EFM can print any fluid that transitions to a solid or semi-solid stateafter deposition. Examples of materials which can be manufactured usingEFM are ceramics, metals, polymers, sol-gel mixtures, composites, andgels. Concretes, cements, clays, slurries and colloids of metals andmetallic or semi-metallic oxides, epoxies, resins, silicones, andthermoplastics can all be assembled using EFM. Additionally, EFM is alsocapable of 3D printing gels made from proteins, polysaccharides, orother polymers and stable hydrocolloids. Additionally, foods such asNutella, mayonnaise, or chocolate, and adhesives such as cyanoacrylatescan also be assembled using EFM. EFM can also be used to printbiologically derived protein and/or polysaccharide hydrogels such aschitosan or Matrigel, synthetic hydrogels such as polyethylene glycol(PEG) based hydrogels, and other synthetic gel, elastomer and rigidpolymers such as polydimethylsiloxane, polyurethanes, thermosets,coacervate solids, and foams.

Support materials used in EFM can include any slurry or fluid exhibitingproperties which allow it to support the embedding of a fusiblematerial. Some examples of support materials are mayonnaise,albumin-foams, gelatin slurries, poly(N-isopropylacrylamide) (PNIPAAM)slurries, polyacrylate slurries, alginate slurries, and structuredfluids displaying non-newtonian, Bingham Plastic behaviors, or otherviscoplastic materials such as Herschel-Bulkley fluid. Conditions insidethe support materials could be used to trigger the transition of theembedded material from fluid to solid or semi-solid state.

For example, a ceramic slurry consisting of an aggregate materialsurrounded by a suspension of hydrating minerals can be deposited into apseudofluid slurry. This enables the printing of cement-like orcolloidal ceramics into complex shapes without the need for a powder &binder-based printing process. Additionally, a Bingham plasticoil-in-water emulsion could be used as a support material.

In some implementations, a 3D printing technique referred to as FreeformReversible Embedding of Suspended Hydrogels (FRESH) to enable the 3Dprinting of soft, biological hydrogel structures that may be too soft tofabricate using other 3D printing techniques. The structures may be any3D design including anatomical structures in humans or the targetorganism for the object, such as the tissue engineering of an organ.FRESH enables true freeform printing of complex geometries and enablesthe geometrically uninhibited fabrication of biopolymers and cellsuspensions by embedding them in thermo-reversible support material.FRESH printing may be a significant improvement over alternative 3Dbioprinting approaches in terms of capability, cost, safety, speed, andease of use. FRESH is cross-platform, and can therefore be easilyadopted for use on an open-source or proprietary FDM 3D Printer.

FRESH uses a support bath material that enables biological hydrogels tobe directly printed in 3D complexity using a range of soft biomaterialsincluding alginate, collagen, hyaluronic acid, and fibrin. Thethermo-reversible support bath may be composed of microparticles thatact as a Bingham plastic or Herschel-Buckley fluid during the printprocess. As a nozzle moves through the bath, it shear thins and offerslittle mechanical resistance, yet the hydrogel being extruded out of thenozzle and deposited within the bath is held in place. The nozzle of theextruder moves through the support material fast enough such that thenozzle generates a shear stress above a threshold shear force andtherefore sees the support material as a fluid. In contrast, thestructure material deposited by the extruder out of the nozzle has ashear stress below the threshold shear force and therefore sees thesupport material as a solid material, and thus stays where it isdeposited. Thus, soft materials that would collapse if printed in air orother bath materials are easily maintained in the intended 3D geometry.The process is performed in a sterile aqueous, buffered environmentcompatible with cells, which means cells can be extruded out of theprinter nozzle with the hydrogel and maintain viability. Once the entire3D structure is FRESH printed, the thermo-reversible property of thesupport bath material can be used to melt out the support bath at acell-friendly 37° C., completely removing the support in anon-destructive manner.

A range of materials using FRESH have been 3D printed, includingalginate, collagen, fibrin, matrigel, photo-crosslinkable hyaluronicacid, cell suspension in collagen, and PDMS, which is a non-hydrogel. Awide range of 3D objects have been printed including geometric solids,vascular networks, whole organs such as the embryonic heart, andintricate scaffolds. FRESH can be used to tissue engineer a range oftissue types, including, for example, cardiac muscle tissue for therepair of congenital heart defects and myocardial infarction, vascularnetworks for the repair/regeneration of skeletal muscle tissue, andhigh-fidelity tissue engineering of skeletal muscle for craniofacialrepair. The possibilities can extend beyond muscular tissues,potentially allowing for any known tissue type, including precursorembryonic tissues. The engineered tissues created using FRESH can beused in vitro models of tissue/organ function and/or as disease modelsystems.

FRESH may be used to 3D print biological tissues and tissue engineeringscaffolds for application in regenerative medicine. A thermoreversible,Bingham plastic slurry supports the embedding, assembly, and release ofa gelling polymer. In turn, conditions inside the slurry could be usedto trigger the polymerization of the printed material. For the slurry,gelatin may be used in a calcium-rich solution, and for the gellingpolymer, alginate may be used. However, FRESH is not limited to thesematerials. FRESH can be expanded to include any Bingham plastic orHerschel-Buckley fluid support material. Additionally, any gellingbiomaterial or organogel can be used as an ink. Depending on the ink,any reversible gel can be used as a support, for example, alginate gels,collagen gels, PNIPAAM, or an organogel.

Using FRESH, perfusable scaffolds of coronary vasculatures can befabricated using public-access MRI data, for example, open-sourcesoftwares such as Skeinforge and ReplicatorG, and an open-sourceMakerBot 3D printer. For example, FRESH can be used to produceanatomically correct, perfusable scaffolds of coronary arteries.Scaffolds with this range of mechanical properties are required for thetissue engineering of soft tissues. In one example, alginate orproteinaceous scaffolds composed of various geometries can be 3D printedin a support material. The support material may be a gelatin slurry inthe form of micronized particulates. The support material is placed in asupport bath supplemented with calcium chloride (CaCl₂). Alginatescaffolds are then 3D printed within the support bath, where the calciumions crosslink the alginate and the gelatin mechanically supports thealginate. Once the alginate scaffolds are printed, the gelatin is meltedby heating the gelatin to its melting temperature (37° C.) and thenremoved by washing it out.

While the above example demonstrates FRESH using alginate crosslinked bycalcium chloride in the support bath, alginate is just one example ofthe embedded material. For example, the alginate can be modified withcovalently grafted polypeptides to add additional biofunctionality suchas adhesion sites for integrins or degradation sites that can be cleavedby matrix metalloproteinases. The alginate can also be blended withother polymers such as collagen or fibrin. Instead of alginate, fibrincan be printed by putting fibrinogen in the extruder and then use thecalcium chloride in the bath and/or thrombin in the support bath tocrosslink the fibrinogen into fibrin. Another example is printingcollagen type I. The collagen I may be in a slightly acidic solution toprevent polymerization. The collagen type I is placed in the syringe andprinted in a gelatin bath. The gelatin bath has a neutral pH, whichtriggers gelation of the collagen I. After printing, the bath is heatedto 37° C., which simultaneously provides additional crosslinking of thecollagen and melts the gelatin. As an alternative to heating,photochemistry may be used to crosslink the embedded material. Forexample, photocrosslinkable hyaluronic acid can be printed in thesupport bath, and UV light applied during the print process and/orafterwards is used to crosslink the hyaluronic acid into a hydrogel.There are multiple comparable examples of embedded soft materials andusing various factors (e.g., heat, enzymes, light) to trigger transitionof the printed material from a fluid into a gel or stiffer materials.Factors that trigger transition of the printed material can include,e.g., chemical exposure, vibration, radiation using ultraviolet,infrared, or visible light, and application of a magnetic or electricfield.

While the above example describes the support bath as a gelatinparticulate slurry that acts as a Bingham plastic-like material, thereare a wide range of other materials that can be used to create thesupport bath. For example, instead of gelatin, ultra-low melting pointAgarose can be used in a similar manner, where particulates are formedand used to make a slurry. The Agarose has a similar thermo-reversiblecharacteristic to gelatin and thus can be melted at physiologictemperatures. Other forms of Agarose with higher melting temperaturescould be used for print materials with different thermal properties.

FRESH printing can be implemented using a 3D printer based onopen-source designs, or commercially available derivatives. For example,a fab@home model 2 3D printer comes with a syringe-based tool that canextrude soft materials. The fab@home is a widely used syringe-basedprinter for soft materials with examples of the printer being used tocreate alginate heart valves, silicone chemical reaction vessels, andeven a bionic ear.

As another example, a RepRap derivative printer, which has a large arrayof open-source tools, is used. The stock MakerBot uses fused filamentfabrication (FFF) where a thermoplastic filament (typically ABS or PLA)is fed into a heated chamber, melted, and then extruded before it coolsand solidifies. This process may not work for hydrogels because they aretoo soft (flexible) to be used in filament extrusion. A syringe extruderis available on the MakerBot, termed the Frostruder, and a variant isused to 3D print sugar scaffolds that could be used to template vascularnetworks. However, the large volume of the syringe and pneumatic controlmay not provide the desired level of control. A syringe-pump basedextruder may be designed on SolidWorks where the stepper motor from theoriginal extruder is used via a direct gear drive to push the plunger ofa syringe with Luer lock tip. The syringe-pump extruder was 3D printedof PLA or ABS plastic using the MakerBot's stock FFF extruder. The FFFextruder was then replaced with the syringe-pump extruder, convertingthe MakerBot into a bioprinter.

FIG. 1 shows a 3D CAD rendering of the syringe-pump extruder 100 showingthe location where the syringe is mounted (syringe holder 102), wherethe support carriage 104 is that mounts to the MakerBot X-axis rails andthe location where the stepper motor is housed (stepper motor housing106). Note that the stock extruder is first used to print thesyringe-pump extruder 100 in PLA or ABS plastic, and then replaced. Theoverall design of the syringe-pump extruder 100 has similar mass andshape to the original stock extruder and once mounted integratesseamlessly with the MakerBot hardware and software. The only calibrationnecessary is to determine the number of motor steps that extrudes agiven amount of fluid, which is determined experimentally for thealginate and collagen hydrogels printed. Typically, it places a 250 μmdiameter stainless steel nozzle on the end of the syringe, but a rangeof sizes can be selected to control the diameter of extruded material.

FIG. 2 shows FRESH printing using a gelatin slurry as thethermo-reversible Bingham plastic support bath 202 and alginate 204 asthe soft hydrogel being printed. The size of gelatin particles mayinterfere with alginate print topology, so gelatin slurries may beblended in a common blender, resulting in a narrower distribution ofsmaller particles. For example, slurries blended for 60 seconds wereshown to have a dominant elastic behavior that is characteristic of aBingham Plastic. CaCl₂ in the support bath 202 provides divalent cationsto crosslink the alginate 204 as it is extruded out of the nozzle 206.The printing process occurs at room temperature (e.g., approximately 20°C.). Layers of deposited alginate gel, each representing a 2-dimensionalslice of a 3D object, were built up by successive depositions to createa 3D scaffold. Once the printing is complete, the temperature isincreased to 37° C. in order to melt the gelatin of the support bath 202and release the embedded alginate structure 208.

Gelatin is selected as the material for the support bath 202 for thebioprinting of soft biological materials because it has many advantagesincluding (i) inexpensive, (ii) biocompatible, (iii) thermo-reversibleat physiologic temperatures, and (iv) readily available in a particulateform. Gelatin is a widely used biopolymer derived from collagen type Iand normally is dissolved in warm water and then cooled down to form acrosslinked hydrogel. In this form, gelatin is not a Bingham plastic andwill not work as the support bath 202. However, the gelatin powder aspurchased can be added to room temperature (approximately 20° C.) water,and then the excess water can be drained off to form a gelatinparticulate slurry. Alternatively, the gelatin slurry can be made byblending a block of gelatin gel in a jar with additional fluid. Theresult is a gelatin slurry that has gelatin particles with lowerstiffness and greater transparency than the block of gelatin gel.However, any manner in which small gelatin gels can be fabricated can beused to make a gelatin slurry. For example, emulsion of warm gelatinsolution in oil followed by cooling results in a gelatin microspheresuspension that can also be used as a support material.

The gelatin slurry behaves as a Bingham plastic, where the hydratedparticulates act as a viscous fluid under the high stress of theextruder nozzle 206 moving through the bath 202 but a solid under lowstress. 3D printing is done at room temperature to keep the gelatin as asolid, and alginate 204 is extruded into the slurry as the printedmaterial. The support bath 202 is supplemented with CaCl₂ to providedivalent cations to crosslink the alginate 204 as it is extruded out ofthe nozzle 206. Once the 3D printing is complete, the bath 202 is heatedto 37° C. in order to melt the gelatin and release the embedded alginatestructure 208.

FRESH can be used to fabricate biological materials and structures. Thegelatin slurry has an aqueous environment with neutral pH andphysiological levels of CaCl₂. The temperature range is cell friendly,with most cells able to survive at room temperature for the printingprocess and at 37° C. for the thermal release. Initial studies printingC2C12 cells inside the alginate has shown greater than 97% cell survivalusing Live/Dead cell staining.

This system can print a large range of 3D objects including basicgeometric solids, various 2D and 3D meshes, and anatomical structuresbased on medical imaging data. For example, FIGS. 3A and 3B show FRESHprinting of the logo 306 using a 3D printer including an XYsyringe-based extruder 300 that deposits hydrogels from a nozzle 302into a thermo-reversible support bath 304. The FRESH printing process isbased on using a thermo-reversible support bath 304 that can be removedafter the printing process is completed. The support bath 304 needs toallow the nozzle 302 of the extruder 300 to move through the bath 304with minimal resistance, but provide support to the extruded hydrogel.The support bath 304 may be achieved using a thermo-reversible Binghamplastic material, which is solid at low stresses and a viscous fluid athigh stresses. The support bath 304 enables the embedding of a hydrogelduring printing followed by non-destructive release when completed. Thethermo-reversible support bath 304 may be composed of microparticlesthat act as a Bingham plastic during the print process.

As the nozzle 302 moves through the bath 304 it shear thins and offerslittle mechanical resistance, yet the hydrogel being extruded out of thenozzle 302 and deposited within the bath 304 is held in place. Thus,soft materials that would collapse if printed in air, or other bathmaterials, are easily maintained in the intended 3D geometry. Once theentire 3D structure is FRESH printed, the thermo-reversible property canbe used to melt out the support bath 304 at a cell-friendly 37° C.,completely removing the support bath 304 in a non-destructive manner.The printing is done in a sterile aqueous, buffered environmentcompatible with cells, which means cells can be extruded out of theprinter nozzle with the hydrogel and maintain viability.

In FIGS. 3A and 3B, the logo 306 was printed in alginate in the gelatinslurry support bath 304. The alginate was mixed with food coloring toaid visualization during the printing process and various time points(a)-(d) show the layer-by-layer fabrication. The cloudiness of the bathis due to the particulate gelatin. As the nozzle of the extruder movesthrough the bath it disrupts the gelatin particles above the printingplane, but not below it. The logo 306 is printed in high fidelity at a 5mm height, and even the overhang of the letter “C” is well formed andperfectly maintained using the support bath 304, which would bedifficult to print with soft materials using other methods. Once theprint is completed, the printing platform of the 3D printer is heatedabove 37° C. in time points (e) and (f) to melt the gelatin so theprinted logo 306 can be removed from the bath 304. Note the surface ofthe bath 304 in the last time point (f) where the change from particlesto liquid is visible.

Similarly, 3D models of the right coronary arterial tree from a humanheart can be scaled-down and printed using FRESH with high fidelity.FIG. 4A shows a 3D CAD model 400 of the coronary artery vasculature froma human heart. Coronary artery 3D models based on whole-body MRI imagingcan be downloaded from a database that has 3D data for major arteriesand veins in the body. The right coronary arterial tree was downloadedand the hollow model 400 was created in MeshLab by resampling the solidmodel to create a smaller child model with inverted normals to serve asnegative space. When the models are flattened as layers into the samemesh file, a hollow model 400 with internal and external surfacesresults.

FIG. 4B shows a scaled-down FRESH printed vasculature 404 embeddedwithin a support bath 406 with a US penny 408 for size reference. Usingfluorescently-labeled alginate, the arterial tree was 3D printed with aninternal luminal diameter of approximately 1 mm. The printed vasculature404 can be released intact from the thermo-reversible support bath 406and imaged using a laser scanning confocal microscope to visualize thehollow lumen that is generated. FIG. 4C shows the FRESH printedvasculature 404 after thermal release from the support bath 406 andimaged using a confocal microscope showing the hollow lumen with adiameter of approximately 1 mm. These scaffolds have been printed with100% alginate a 50:50 mixture of alginate and collagen, pure collagen 1solution, and pure fibrinogen solution.

Tubular and beam-like structures with simple mechanical behaviors can becompounded to print constructs with higher order functions. For example,solids with negative Poisson's ratios or hollow geometries can beprinted. FIG. 5 shows a printed alginate mesh of collapsed hexagonalunits in an unstrained form 500 and a strained form 502 under axialstrain displaying transverse expansion, which indicates a negativePoisson's Ratio. FIG. 6 shows a parametric tubular structure 602 with a2 mm inner diameter and bifurcation that was designed in CAD andprinted, revealing a gel manifold capable of dividing fluid flow. FIG. 7shows a cross-section view 702, a side view 704, and a close-upcross-section view 706 of a two-material cylinder with inner material710 and outer material 712. The cross-section view 702 and the side view704 are shown at a scale of 2 mm. The close-up cross-section view 706 isshown at a scale of 500 μm.

Another example of a structure that can be printed using FRESH is afilament. FIG. 8 shows a fluorescence image 800 of an alginate filament802 embedded among gelatin particles 804 at a scale of 1 mm. FIG. 9shows a rectilinear pattern 900 of filaments 906 and fluorescence images902 and 904 of the filaments 906 at a scale of 500 μm. FIG. 10 shows anoctagonal pattern 1000 of filaments 1006 and fluorescence images 1002and 1004 of the filaments 1006 at a scale of 500 μm.

Yet another example of a structure that can be printed using FRESH is ahelix. FIG. 11 shows a helix 1010 being printed at various time points(a)-(c). FIG. 11 shows that FRESH can be used to print a 3D structure inany direction in 3D space, including in a non-planar fashion.

While alginate is a suitable print material for some structures, othermaterials may be used to print biological materials and structures. Ingeneral, alginate is not cell adhesive, and therefore may not besuitable for use in printing tissue engineering scaffold. One option isto modify the alginate with cell adhesive polypeptides of proteins. Forexample, alginate can be modified by the covalent linking of thearginine-glycine-aspartic acid (RGD) amino acid sequence to bind cells,or a wide range of other bioactive molecule. This may expand the utilityof the alginate directly as a biomaterial and the optimal concentrationof RGD-modified alginate will need to be added in order to make theprinted alginate cell adhesive. The alginate can be used to facilitategelation of other hydrogel biomaterials. Specifically, blends ofalginate may be developed with both collagen type I and fibrinogen inorder to develop cell adhesive scaffolds that can be remodeled by cells.

FRESH can be used to 3D print PDMS. The PDMS is printed as a liquid thatcures over 48 hours. FIG. 12 shows 3D printed PDMS in the form ofinterconnected chain links 1012.

FRESH demonstrates the application of fused deposition principles to the3D printing of a gelling polymer. It utilizes widely-availableopen-source software and hardware to create a functional gel-prototypingsystem with true freeform fabrication capabilities. Gelatin slurry is abiocompatible, inexpensive, and readily available Bingham Plastic whichliquefies just below human body temperature. Initial prints using thepresent invention can be done with food-grade gelatin powder retrievedfrom various grocery stores. Alginate is a biocompatible, inexpensive,readily available, and reversible material for cell-encapsulation.Alginate can also support the gelation of major biopolymers such ascollagen and fibrin. Thus, FRESH may enable true freeform fabrication ofany gelling biopolymer. Any biopolymer like collagen can be mixed andFRESH-printed with alginate; the alginate can be removed to reveal acollagen print. FRESH can be implemented on any RepRap 3D printer.Furthermore, these printers can be modified to print complex gelstructures and perfusable vascular models with biocompatible materials.

FRESH is designed to enable rapid tissue prototyping and fosterwidespread iterative improvements to engineered tissue designs. FRESH iscapable of 3D printing any hydrophilic gel as long as it can be confinedby a mechanism such as diffusion limiting. The gelatin support slurrycan be combined with cell growth medium and gelling biopolymers such ascollagen and fibrin, and Matrigel can be printed with cells toeffectively 3D print living tissues. In one example, live-dead assaysdemonstrate high cell viability during FRESH printing with Collagen Iand C2C12 cells. FIG. 13 shows an image 1100 and a 3D representation1102 of C2C12 cells printed in Collagen I gel. Image 1100 shows theconcentration of cells 2 hours after printing. The 3D representation1102 shows the concentration of cells in the collagen construct 24 hoursafter printing. As seen in the 3D representation 1102, the cells attachand spread throughout the printed collagen construct. FIG. 14 shows 3Drepresentations 1200, 1202, 1204 of C2C12 myoblasts in a collagenconstruct. The 3D representations 1200 and 1202 show the concentrationof cells after 24 hours of incubation, and the 3D representation 1204shows the concentration of cells after 7 days of incubation. The 3Drepresentations 1202 and 1204 are shown at a scale of 50 μm. FIG. 15shows 3D representations 1300, 1302, 1304 of MC3T3 fibroblasts in acollagen construct. The 3D representations 1300 and 1302 show theconcentration of cells after 24 hours of incubation, and the 3Drepresentation 1304 shows the concentration of cells after 7 days ofincubation. The 3D representations 1302 and 1304 are shown at a scale of50 μm. The 3D representations of the cells in the collagen constructsshow that cells spread quickly and reach high density as the tissuecompacts and the cells multiply. Because a tissue printed using FRESHcan be released by placement in an incubator running at 37° C., FRESHessentially represents a two-step solution for rapidly prototypingtissues. Open-source frameworks for cell incubation are actively beingdeveloped, and a one-step print-and-incubate Rapid Tissue Prototypingmachine is plausible.

FIG. 16 is a flowchart of a process 1400 for fabricating amultidimensional structure. Briefly, the process 1400 includes providingsupport material within which the structure is fabricated (1402);depositing, into the support material, structure material to form thefabricated structure (1404); treating the structure material to causethe structure material to transition from the fluid to the solid orsemi-solid state after the deposition of the structure material (1406);and removing the support material to release the fabricated structurefrom the support material (1408). The process 1400 will now be describedin the context of FRESH printing alginate scaffolds in a gelatin slurrybath. However, other suitable materials may be used instead of alginateand gelatin.

A support material, such as a gelatin slurry bath, is provided (1402).To create the gelatin slurry bath, 30 grams of gelatin may be hydratedin excess 7 mM CaCl₂ at 4° C. The slurry may be blended with a blenderfor 60 seconds in a 500 mL mason jar and centrifuged at 1400 rpm for 3minutes. Slurry supernatant may be aspirated and replaced with 7 mMCaCl₂ at 20° C. The slurry may be resuspended by vortexing. This washingprocedure may be repeated two additional times, except the gelatinslurry is not replenished with 7 mM CaCl₂ the final time. Washed slurrymay be loaded into 60 mm diameter glass petri dishes to be used forFRESH prints.

The structure material may be a solution of fluorescent alginate. Asolution of 2.0%-w/v Sodium Alginate, 0.02%-w/v 6-Aminofluorescein,0.022%-w/v EDC, and 0.025% w/v Sulfo-NHS may be prepared and stirred for48 hours at 20° C. Unreacted 6-Aminofluorescein may be removed by fiveconsecutive 12 hr dialysis shifts against 2%-w/v Sodium Alginate at 4°C. in dialysis cassettes.

Before printing, a standard 3 mL syringe with a 0.25 mm-ID stainlesssteel nozzle may be filled with a solution of 0.08%-w/vfluorescein-labeled alginic acid and 1.92% w/v Sodium Alginate andloaded onto a custom-modified Replicator 3D Printer. Digital models ofthe alginate scaffolds may be created using, for example, SolidWorks andMeshLab softwares. Alginate scaffolds were printed in the gelatin slurrybath at 20° C. (1404), heated to 37° C. (1406), and washed free frommolten gelatin slurry with 0.13 M CaCl₂ (1408).

FRESH makes it possible to 3D print protein scaffolds within athermally-reversible support bath that can be selectively remove oncethe entire scaffold is complete. Thus, FRESH can be used to engineer avariety of tissues.

For example, FRESH can be used to engineer complex 3D heart tissue witha level of structure and functional performance that is difficult tocreate using alternative approaches. To 3D print anatomically-optimizedengineered cardiac tissues, a 3D computer model of the neonatal humanventricle may be built based on 3D MRI scans, which enables the use ofcomputational modeling to optimize the tissue design to maximizeperformance as well as optimize the design for 3D printing. An enhancedversion of the FRESH 3D printing process is used that usesalginate-collagen blends for fabricating tissue engineering scaffolds.Printing parameters to maximize cell survival and integration into theconstruct are optimized, making sure to match degradation rates with theformation of new muscle tissue. A 3D human heart muscle may then beengineered using human stem cell derived cardiomyocytes. FIG. 17 shows aFRESH print of an embryonic chicken heart 1500 next to a penny 1502 anda fluorescence image 1504 of a layer of the FRESH printed embryonicchicken heart 1500 at a scale of 1 cm. The fluorescence image 1504 showsthe internal structures of the FRESH printed embryonic chicken heart1500.

As another example, FRESH can be used for 3D printing of complexvasculature for repair of traumatic muscle injury. FRESH 3D bioprintingcan be used in order to engineer patient-specific vascular networks forrapid regeneration of large-volume muscle defects. This vascular networkmay be composed of ECM proteins and serves as the scaffold to guidemuscle regeneration as well as provide nutrient mass transport. FRESHcan engineer patent, complex, 3D vascular networks that scale from mainarteries and veins down to arterioles and venuoles, with 1 mm internaldiameter. This level of structural and functional performance may bedifficult to create using alternative approaches. FRESH has thecapability to 3D print a high-density vascular network, which can beused to engineer large-volume skeletal muscle constructs.

FRESH can be used to engineer high-fidelity muscle tissue engineeringfor craniofacial repair. Fine skeletal muscles appropriate forcraniofacial reconstruction following traumatic injury to the head canbe engineered. For example, skeletal muscle cells can be integrated intonanostructured fibers composed of the extracellular matrix protein, suchas collagen. This will result in a living cell thread composed entirelyof cells and biopolymers, eliminating any synthetic polymers from thesystem. Skeletal myoblasts can be seeded onto collagen fibers 3D printedwith FRESH, or mixed with the collagen and FRESH printed directly ascell-laden fiber to create living muscle fiber threads of highly alignedmuscle cells. Once myoblasts cell fibers are created, the cells willalign and fuse into myotubes. Once the living muscle fiber threads aregenerated with FRESH, they can be used to weave muscle tissue constructsthat recapitulate the overall shape and cellular architecture ofcraniofacial muscles. Using custom bioreactors, support posts can beintegrated into 35-mm cell culture Petri dishes around which the cellthreads can be weaved. Alternatively, the posts and cell threads can beprinted in the same construct. The posts can be printed from PDMS, andthe tissue can be printed from collagen and cell suspensions. This wouldbe an example of printing hybrid synthetic and living prints withcomplex purposes. By proper placement of these post and weavingpatterns, a variety of muscle structures can be regenerated.

FIG. 18 illustrates weaving used to regenerate various muscle structuresusing the living muscle fiber threads. FRESH provides the capability toregenerate both parallel muscle tissue types 1602 and convergent muscletissue types 1604. Parallel muscles 1602 have a spindle shape andcircular cross-section with all muscle cells aligned in the same,uniaxial direction. To recreate this structure, the living muscle fiberthread 1607 is wrapped back-and-forth between to support posts 1606 and1608. Convergent muscles 1604 have more of a fan shape correspondingcross-section that is thin and wide. To recreate this structure, theliving muscle fiber thread 1610 is wrapped around one post 1612 on theright and then a tightly spaced column of posts 1614 on the left.Alternatively, FRESH can be used to print the muscle fibers directlyinto the parallel and convergent muscle structures with or without thesupport posts. Other muscle structures that can be engineered usingFRESH include circular muscle bundles such as sphincters appropriate forrepair of the orbicularis oculi muscle around the eye, and theorbicularis oris muscle around the mouth.

A number of implementations have been described. Nevertheless, variousmodifications can be made without departing from the spirit and scope ofthe processes and techniques described herein. In addition, theprocesses depicted in the figures do not require the particular ordershown, or sequential order, to achieve desirable results. In addition,other steps can be provided, or steps can be eliminated, from thedescribed processes, and other components can be added to, or removedfrom, the describe apparatus and systems. Accordingly, other embodimentsare within the scope of the following claims.

What is claimed is:
 1. A method for fabricating a structure having atleast two dimensions, the method comprising: providing support materialwithin which the structure is fabricated; wherein the provided supportmaterial is stationary at an applied stress level below a thresholdstress level and flows at an applied stress level at or above thethreshold stress level during fabrication of the structure; and whereinthe provided support material is configured to mechanically support atleast a portion of the structure and to prevent deformation of thestructure during the fabrication of the structure; depositing, into thesupport material, structure material to form the fabricated structure;wherein the deposited structure material is suspended in the supportmaterial at a location where the structure material is deposited; andwherein the structure material comprises a fluid that transitions to asolid or semi-solid state after deposition of the structure material;and removing the support material to release the fabricated structurefrom the support material.
 2. The method of claim 1, wherein the supportmaterial comprises a gel material.
 3. The method of claim 1, wherein thesupport material comprises a hydrogel material.
 4. The method of claim1, wherein the support material comprises micronized particulates. 5.The method of claim 1, wherein the support material comprises athermo-reversible material.
 6. The method of claim 1, wherein thestructure material comprises a material having an elastic moduli of lessthan 1 megapascal.
 7. The method of claim 1, wherein the structurematerial comprises at least one of a gel material that is different fromthe support material, a metal material, a ceramic material, or a polymermaterial.
 8. The method of claim 1, wherein the structure materialcomprises at least one of an alginate material, a collagen material, afibrin material, a hyaluronic acid material, a protein material, apolysaccharide hydrogel material, synthetic gel material, an elastomerpolymer material, a rigid polymer material, or a polydimethylsiloxane(PDMS) elastomer.
 9. The method of claim 1, wherein the structurematerial is deposited using a syringe-based extruder that is insertedinto the support material and extrudes the structure material into thesupport material.
 10. The method of claim 1, further comprising:treating the structure material to cause the structure material totransition from the fluid to the solid or semi-solid state after thedeposition of the structure material.
 11. The method of claim 10,wherein the support material comprises a crosslinking agent for treatingthe structure material to cause the structure material to transitionfrom the fluid to the solid or semi-solid state after the deposition ofthe structure material.
 12. The method of claim 11, wherein thecrosslinking agent comprises at least one of calcium chloride orthrombin.
 13. The method of claim 10, wherein the support materialcomprises a material having a different pH from the structure materialfor treating the structure material to cause the structure material totransition from the fluid to the solid or semi-solid state after thedeposition of the structure material.
 14. The method of claim 10,wherein treating the structure material comprises heating the structurematerial.
 15. The method of claim 10, wherein treating the structurematerial comprises cooling the structure material.
 16. The method ofclaim 10, wherein treating the structure material comprises radiatingthe structure material with ultraviolet light.
 17. The method of claim1, wherein removing the support material comprises heating the supportmaterial.
 18. The method of claim 1, wherein removing the supportmaterial comprises cooling the support material.
 19. The method of claim1, wherein removing the support material comprises removing cations todisrupt crosslinking of the support material.
 20. The method of claim 1,wherein the fabricated structure is a biological tissue or a tissueengineering scaffold.
 21. The method of claim 1, wherein depositing,into the support material, the structure material to form the fabricatedstructure comprises: depositing the structure material layer by layer inan XY plane.
 22. The method of claim 1, wherein depositing, into thesupport material, the structure material to form the fabricatedstructure comprises: depositing the structure material layer by layer inan XZ plane.
 23. The method of claim 1, wherein depositing, into thesupport material, the structure material to form the fabricatedstructure comprises: depositing the structure material in a non-planarconfiguration.
 24. The method of claim 1, wherein the fabricatedstructured has three-dimensions, and wherein the fabricated structurehas printed anisotropic mechanical properties in a direction that lie ina XY plane.
 25. A method for fabricating a tissue scaffold, the methodcomprising: performing a printing operation at approximately twentydegrees Celsius, the printing operation comprising: providing a gelatinslurry support bath within which the tissue scaffold is fabricated;wherein the gelatin support bath flows in a presence of an applied forceand transforms into a non-flowing fluid in an absence of the appliedforce; extruding, from a nozzle, a hydrogel into the gelatin slurrysupport bath to form the tissue scaffold, with the hydrogel having anelastic moduli of less than 1,000 kilopascal; wherein the gelatin slurrysupport bath includes calcium chloride to provide divalent cations tocrosslink the hydrogel as the hydrogel is extruded out of the nozzle;and wherein the gelatin slurry support bath is configured tomechanically support the hydrogel and to prevent deformation of thehydrogel during printing of the tissue scaffold; and placing the gelatinslurry support bath with the extruded hydrogel into an incubator that isheated to approximately thirty-seven degrees Celsius to melt the gelatinslurry support bath and to release the tissue scaffold.
 26. The methodof claim 25, wherein the hydrogel comprises at least one of alginate,collagen type I, fibrin, or hyaluronic acid.
 27. The method of claim 25,wherein the hydrogel comprises a cell laden hydrogel.